Paul W. Singleton

Enrichment of Bradyrhizobium spp populations in soil due to cropping of the homologous host legume

Host legumes can enrich their immediate soil environment with rhizobia through rhizosphere effects. The extent to which this enrichment occurs, the specificity of the process and its interaction with soil management factors remain poorly described. In a series of field trials, we measured changes in the size of indigenous populations of Bradyrhizobium in response to cropping of host and non-host legumes under two N fertilizer regimes. Uninoculated cowpea (Vigna unguiculata) and soybean (Glycine max) were grown with or without applied urea (900 kg N ha−1) at three field sites on the island of Maui, HI, U.S.A., not previously cropped with legumes. Using the most-probable-number plant infection method and Siratro (Macroptilium atropurpureum) as the host, the population density of Bradyrhizobium in the bulk soil at each site was measured at planting and at grain maturity and compared to the population density in adjacent fallow soil. When the size of the initial indigenous population was low (18 and 580 cells g−1 soil), significant increases in the population density compared to fallow soil were observed only in soils cropped with cowpea receiving no applied urea. When the size of the initial indigenous bradyrhizobial population was high (5.8 × 104 cells g−1 soil), no significant increase in the population density was observed. These results suggested that enrichment of soil bradyrhizobial populations was host-specific, that symbiotic legumes can enrich their soil environment with microsymbionts up to a threshold level and that such enrichment can be curtailed by soil management practices that suppress nodulation.

Bradyrhizobium spp. (TGx) isolates nodulating the new soybean cultivars in Africa are diverse and distinct from bradyrhizobia that nodulate North American soybeans

The newly developed cultivars of soybean in Africa, known as Tropical Glycine cross (TGx), are nodulated by bradyrhizobia indigenous to African soils, here designated Bradyrhizobium spp. (TGx). Isolates of Bradyrhizobium spp. (TGx) obtained from nodules of TGx soybeans that were inoculated with soils from 65 locations in six African countries were characterized and grouped into 11 phylogenetic clusters on the basis of RFLP of the 16S rRNA gene. Five restriction enzymes (RsaI, HinfI, MspI, CfoI and HaeIII) established RFLP groups within these Bradyrhizobium spp. (TGx) isolates, which were used to construct a phylogenetic tree showing their genetic relationship with other Bradyrhizobium species. RFLP analysis indicated that Bradyrhizobium spp. (TGx) is a heterogeneous group with some isolates related to Bradyrhizobium japonicum and Bradyrhizobium elkanii strains and some to Bradyrhizobium spp. (misc.) reference strains isolated from a variety of tropical legumes. The heterogeneity within the large phylogenetic clusters was further examined through analysis of randomly amplified polymorphic DNA (RAPD) using GC-rich PCR primers. The RAPD analysis showed additional heterogeneity in the Bradyrhizobium spp. (TGx) phylogenetic clusters, which was not revealed by separations based on RFLP analysis. The Bradyrhizobium spp. (TGx) isolates were classified into effective and ineffective types based on their symbiotic performance on TGx soybean. The isolates were randomly distributed throughout the phylogenetic clusters regardless of their symbiotic effectiveness on TGx soybean.

PHENOLOGY, GROWTH, AND YIELD OF FIELD-GROWN SOYBEAN AND BUSH BEAN AS A FUNCTION OF VARYING MODES OF N NUTRITION

Abstract In field trials conducted at four sites in Hawaii, soybean (Glycine max) and bush bean (Phaseolus vulgaris) were either inoculated with homologous rhizobia, fertilized at high rates with urea, or left unamended. Crop phenology was assessed every few days. Rates of biomass and N accumulation and components of yield were measured five times during each crop cycle to assess the extent to which: (i) crops relying on soil, symbiotic, or fertilizer N differed in their growth characteristics; (ii) mode of N nutrition affected the timing of developmental stages; and (iii) effects of N nutrition on crop growth and development were related to final yield. While all measured variables differed significantly between sites, the effect of changing N source on these variables, in N limited environments, was consistent across sites. Rate and extent of node production, crop growth and yield were increased in symbiotic and N-fertilized crops as compared to unamended, non-fixing crops, while reproductive development was protracted. Extended time required to reach reproductive maturity was attributable to an increase in seed fill duration as time to flowering was not affected. Development and yield of N2-fixing crops were similar but not equivalent to those of N-fertilized crops. To produce reliable yield estimates, legume growth simulation models must be able to accurately simulate crop growth and phenology. The present data indicate that information relating to source and supply of N must be incorporated before such models can be used to generate reliable yield estimations. Results of these trials also provide a valuable dataset for calibrating model subroutines for inorganic nitrogen uptake and nitrogen fixation in soybean and bush bean growth under field conditions and adjusting model coefficients for tropical environments.

Comparison of molecular and antibiotic resistance profile methods for the population analysis of Bradyrhizobium spp. (TGx) isolates that nodulate the new TGx soybean cultivars in Africa

Aims: Comparison of molecular and antibiotic resistance profile methods to identify an easy method that can differentiate between strains of introduced Bradyrhizobium japonicum and the indigenous Bradyrhizobium spp. (TGx) isolates which nodulate the newly developed TGx soybean cultivars in Africa.

Influence of acid soil on nodulation and interstrain competitiveness in relation to tannin concentrations in seeds and roots of Phaseolus vulgaris

Abstract The relationship between acid soils and (1) early nodulation in common beans, (2) interstrain competitiveness of Rhizobium leguminosarum bv. phaseoli strains and (3) tannin concentration in roots, were evaluated in two experiments. Ten Phaseolus vulgaris cultivars of different seed tannin content were grown in pots in a greenhouse in soil at pH 4.5 or 5.5, inoculated with equal amounts of strains CIAT 899 and KIM5. At pH 5.5, on all cultivars strain KIM5 occuppied a greater proportion of nodules than strain CIAT 899. This was reversed at pH4.5. Nodule number and nodule weight, 21 days after inoculation, were strongly reduced but root tannin concentrations doubled at pH 4.5. Nodule nmbers were negatively correlated with tannin concentration in roots at both pH values. Seed tannin content was negatively correlated with early nodulation, but this was statistically significant only at pH 5.5. Nodulation of cultivars with high seed tannin contents was generally more acid tolerant than of those with low seed tannin content. In a second experiment, six Phaseolus vulgaris cultivars were planted at two field sites with acid soil and one site with neutral soil. In the field, as in the pots, nodule numbers at early stages of plant development were reduced by 30–50% in the acid soils compared to the neutral soil. In contrast, root tannin content in acid soils was twice that in the neutral soil. There were significant cultivar and cultivar by location effects. Strains of rhizobia were tested for tolerance to bean seed tannin extracts, to seed diffusates and for survival on seed surfaces. The Rhizobium strains were sensitive to tannine extracts, with obvious strain differences. Sensitivity of most strains was greater at acidic pH. Seed diffusates, in contrast, did not inhibit growth of most strains.

The use of nitrogen-15 natural abundance and nitrogen yield of non-nodulating isolines to estimate nitrogen fixation by soybeans (Glycine max L.) across three elevations

SummaryDissimilarities in soil N uptake between N2-fixing and reference non-N2-fixing plants can lead to inaccurate N2 fixation estimates by N difference and 15N enrichment methods. The natural 15N abundance (δ 15N) method relies on a stabilized soil 15N pool and may provide reliable estimates of N2 fixation. Estimates based on the δ 15N and differences in N yield of nodulating and non-nodulating isolines of soybean were compared in this study. Five soybeans from maturity groups 00, IV, VI, and VIII and their respective non-nodulating isolines were grown at three elevations differing in ambient temperature and soil N availability. Despite large differences in phenological development and N yield between the non-nodulating isolines, the δ 15N values measured on seeds were relatively constant within a site. The δ 15N method consistently produced lower N2 fixation estimates than the N difference method, but only in three of the 15 observations did they differ significantly. The average crop N derived from N2 fixation across sites and maturity groups was 81% by N difference compared to 71% by δ 15N. The magnitude of difference between the two methods increased with increasing proportions of N derived from N2 fixation. These differences between the two methods were not related to differences in total N across sites or genotypes. The low N2 fixation estimates based on δ 15N might indicate that the nodulating isolines had assimilated more soil N than the non-nodulating ones. A lower variance indicated that the estimates by N difference using non-nodulating isolines were more precise than those by δ 15N. Since the differences between the estimates were large only at high N2 fixation levels (low soil N availability), either method may be used in most situations when a non-nodulating isoline is used as the reference plant. The δ 15N method may have a comparative advantage over N difference and 15N enrichment methods in the absence of a suitable non-N2-fixing reference plant such as a non-nodulating isoline.

Time-course of dinitrogen fixation of promiscuous soybean cultivars measured by the isotope dilution method

Abstract Soybean cultivars capable of nodulating with indigenous Bradyrhizobium spp. have been developed by the International Institute of Tropical Agriculture (IITA) and national programs in Africa in order to avoid artificial inoculation by resource-poor farmers in Africa. The current selection procedure for enhanced N2 fixation is based on an assessment of nodule formation which does not directly quantify the proportions of crop N derived from the atmosphere. We have monitored N accumulation patterns and N2 fixation in nine promiscuous soybean cultivars with different maturity periods, using the 15N dilution technique. Nodule development generally peaked at the early podfill stage for all cultivars except Tgx 1519-1D and Tgx 1447-2D in which it continued to increase. The proportion of crop N derived from fixation (%NDFA) ranged between 51% and 67%, 77% and 84%, and 66% and 73% at full bloom, early podfill, and physiological maturity stages, respectively. Total N accumulation increased in all soybean genotypes with increasing plant age. Significant correlations (P<0.001) were established between nodule weight and %NDFA, even though this did not explain the relationship between nodule development and N2 fixation in cultivars such as Tgx 1519-1D. Promiscuous soybean cultivars retained between 10% and 19% of total N accumulated at the final harvest, in belowground biomass. Our results indicated that these soybean cultivars can derive substantial proportions of plant N from N2 fixation in soils where compatible indigenous bradyrhizobia populations are adequate and effective. Also, we have substantiated the claims that qualitative nodulation parameters currently used to select varieties with a high N2 fixation capacity need to be validated with other measurements of N2 fixation.

Effectiveness of Sesbania rostrata and Phaseolus calcaratus as green manure for upland rice grown in acidic soil

Two field experiments on green manuring were conducted under upland acidic soil (pH = 4.35) conditions with the following objectives: (1) to determine the influence of inoculation site, P fertilization, and liming on the biomass production, N content, N accumulation, and N availability of S. rostrata grown in an acidic soil, (2) to compare the effectiveness of S. rostrata, P. calcaratus and urea as N sources for upland rice as affected by liming and N source-sowing time combination, and (3) to assess the effect of liming and N source-sowing time combination on % Ndff (N derived from the fertilizer), % Ndfs (N derived from soil), % FNU (fertilizer N utilization), and FNY or fertilizer N yield (kg N ha−1) of upland rice grown in acidic soil. At 2 weeks after incorporating S. rostrata (95 days after lime application), liming significantly increased N availability by more than 2-fold suggesting that the decomposition of S. rostrata by soil microflora was stimulated by lime. Liming, phosphorus application, and inoculation site improved significantly the dry biomass production, N content and N accumulation of S. rostrata; thus, enhancing its green manuring potential. Regardless of liming, S. rostrata whether applied at 0 week or 2 weeks before sowing was superior to urea in improving grain and straw yields. P. calcaratus when applied at 2 weeks before sowing also produced higher grain yield than urea. Immediate sowing of upland rice after green manure incorporation did not affect negatively the growth and development of upland rice; hence, farmers could save at least 2 weeks in their cropping calendar. N source-sowing time combination had a highly significant influence on % Ndff, % Ndfs, % FNU, N uptake, and fertilizer N yield of upland rice. However, only N uptake was influenced significantly by liming. The rice plant obtained significantly higher % Ndfs from the soils treated with green manure than those treated with urea regardless of liming. The % FNU and % Ndff from the green manures were 11-37% and 9-25%, respectively. These values are much lower than those obtained under continuously flooded soil conditions possibly because of the differences in the organic matter decomposer populations and N loss mechanisms between sloping upland conditions and continuously flooded conditions.

Carbon isotope composition of N2-fixing and N-fertilized legumes along an elevational gradient

Dinitrogen-fixing legumes are frequently assumed to be less water-use efficient than plants utilizing soil mineral N, because of the high respiratory requirements for driving N2 fixation. However, since respiration is assumed not to discriminate against 13C, any differences in water-use efficiency exclusively due to respiration should not be apparent in carbon isotope discrimination (Δ) values. Our objective was to determine if the source of N (N2 fixation versus soil N) had any effect on Δ of field-grown grain legumes grown at different elevations. Four legume species, Glycine max, Phaseolus lunatus, P. vulgaris, and Vigna unguiculata, were grown on five field sites spanning a 633 m elevational gradient on the island of Maui, Hawaii. The legumes were either inoculated with a mixture of three effective strains of rhizobia or fertilized weekly with urea at 100 kg N ha-1 in an attempt to completely suppress symbiotic N2-fixing activity. In 14 of 20 analyses of stover and 12 of 15 analyses of seed Δ values were significantly higher (p=0.10) in the inoculated plants than the N-fertilized plants. Nitrogen concentrations were generally higher in the fertilized treatments than the inoculated treatments. The different Δ values obtained depending on N-source may have implications in using Δ as an indicator of water-use efficiency or yield potential of legumes.

Distribution and Characteristics of Bradyrhizobium Spp. Nodulating African Soybeans

The size and effectiveness of indigenous rhizobia populations influence the legume yield response to inoculation. Some African soybean varieties (e.g. TGx lines) were developed to nodulate with indigenous Bradyrhizobium spp. as well as B. japonicum ostensibly to eliminate the need for inoculation with B. japonicum. We characterized the bradyrhizobia populations from 70 soils in 9 African countries according to nodulation phenotypes: B. spp. effectively nodulate cowpea, B. spp. (TGx) effectively nodulate TGx soybean and cowpea, B. japonicum nodulates N. American and TGx soybean and cowpea. Populations of B spp.(TGx) and B. japonicum were ≤102 g-1 in 85% and 91% of the soils indicating inoculation should increase soybean yields. B spp.(TGx) and B. japonicum were not detected in 26% and 67% of the soil samples. Population size was not related to soil physiochemical properties but was related to prior management. B. spp.(TGx) and B. japonicum populations≥103 g -1 soil were more frequent at research stations than farmers’ fields and where soybean had previously been grown. Of 258 isolates we made using TGx soybean as a trap host, only 27% were highly effective on TGx soybean. Most of the effective isolates were also effective on N. American soybean. RFLP analysis of 16S rDNA from B. spp.(TGx), B. japonicum, and B. spp. strains showed that most B. spp.(TGx) strains were phylogenetically related to B. spp. Like B. elkanii strains, B. spp.(TGx) strains do not have the nodY gene. Additional genetic analyses, evaluation of cross inoculation with tropical legumes and IAR analysis indicated the B. spp.